Production of poly-beta-hydroxybutyrate in transformed escherichia coli

ABSTRACT

Methods are provided for enhancing the production of PHB from a transformed E. coli host which includes the genes coding for the PHB biosynthetic pathway. By inserting the genes coding for PHB into a host which includes a lactose utilization system, a low cost minimal medium including whey can be used as the fuel and carbon source for PHB production. A plasmid which codes for the PHB biosynthetic pathway plus four hundred extra bases on either side of the first and last genes in the pathway has been inserted into the host and has been shown to produce a larger amount of PHB accumulation in a shorter period of time than other plasmid constructs. CaCl 2  has been shown to be an effective agglomerating agent for agglomerating PHB which has been produced in a transformed E. coli host.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is related to the following co-pending patentapplication which is herein incorporated by reference:

"Cloning and Expression in Escherichia coli of the Alcaligenes eutrophusH16 Poly-Beta-Hydroxybutyrate Biosynthetic Pathway", of Douglas E.Dennis, which has Ser. No. 07/362,514 and was filed in the Patent andTrademark Office on Jun. 7, 1989.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is generally related to the production ofpoly-beta-hydroxybutyrate (PHB) using Escherichia coli (E. coli) whichhas been genetically transformed by a vector carrying the genes codingfor the PHB biosynthetic pathway and, more particularly, to the moreefficient production of PHB in transformed E. coli.

2. Description of the Prior Art

PHB is an energy storage material produced by a variety of bacteria inresponse to environmental stress and is a homopolymer ofD-(-)-3-hydroxybutyrate which has properties comparable topolypropylene. Because PHB is biodegradable, there is considerableinterest in using PHB for packaging purposes as opposed to other plasticmaterials in order to reduce the environmental impact of human garbage.PHB also has utility in antibiotics, drug delivery, medical suture andbone replacement applications. PHB is commercially produced fromAlcaligenes eutrophus (A. eutrophus) and sold under the tradenameBiopol.

As described in the above incorporated patent application and in thearticle by Slater et al., "Cloning and Expression in Escherichia coli ofthe Alcaligenes eutrophus H16 Poly-β-Hydroxybutyrate BiosyntheticPathway", Journal of Bacteriology, Vol. 170, No. 10, Oct. 1988, p.4431-4436, which is also herein incorporated by reference, it was shownthat E. coli could be genetically transformed with genes from A.eutrophus which code for the PHB biosynthetic pathway. E. coli are a farbetter vehicle for producing PHB than A. eutrophus since more is knownabout handling the bacteria, E. coli, i.e., E. coli is more easilycontrolled and manipulated. The transformed E. coli were able to expressPHB in relatively large quantities.

Despite PHB's advantages over other materials, its high cost ofproduction has hindered its performance in the market. Currently, PHB isproduced in transformed E. coli by growing the E. coli on luria broth(LB) and using glucose as the carbon source. Approximately one third ofthe production cost of PHB is attributable to the cost of the rich LBmedium and the glucose. If a less expensive carbon source could beutilized, the overall cost of PHB production could be significantlyreduced. In addition, much of the total cost of PHB production isattributable to purifying the PHB produced in the E. coli. Currently,PHB is purified by centrifugation, followed by mechanical lysis of thecells to release PHB, a high temperature procedure to agglomerate thePHB, and finally a spray drying step to procure the purified granules.If a less expensive method were available for collecting the PHB fromthe E. coli the overall cost of PHB production could be significantlyreduced.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide improvedtechniques for producing PHB in transformed E. coli.

It is another object of this invention to provide a transformed E. colistrain which can accumulate PHB at higher levels than previous E. colistrains and which can utilize minimal medium containing whey for growingconditions.

It is yet another object of this invention to provide a method ofagglomerating PHB granules from lysed E. coli cells using an ionicsolution.

According to the invention, a strain of E. coli, i.e., E. coli HMS174,has been transformed by a vector containing a plasmid with the PHBbiosynthetic pathway and approximately four hundred extra bases on boththe upstream and downstream sides of the pathway. The HMS174 strain ofE. coli was chosen because it contains a lactose utilization system andis recombination deficient so that a plasmid containing lactose geneticregions will not recombine and make the construct unstable. The lactoseutilization system present in E. coli HMS174 has allowed whey to be usedas a carbon source for the production of PHB. Whey is a waste productfrom cheese processing and is very inexpensive. Experiments have beenperformed which show that the strain of transformed E. coli grows inminimal medium containing whey and has an average yield of PHB ofapproximately 85% (PHB dry weight/total cell dry weight).

In addition, experiments have been conducted which show that PHBproduced in transformed E. coli may be agglomerated with various ionicsolutions. To retrieve purified PHB in large quantities, the transformedE. coli cells are first lysed by mechanical or physical means, such asby sonication, or by genetic means. Then, the cells are incubated in anionic solution, such as 10 millimolar (mM) calcium chloride (CaCl₂),which agglomerates the PHB granules. Finally, the agglomerates arecentrifuged from the culture at low speed. Experiments show that nearlyall (100%) of the PHB in the culture is agglomerated and recovered bythis process. The results are especially exciting since the same type ofagglomeration is not possible for retrieving PHB from A. eutrophus.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects and advantages will be betterunderstood from the following detailed description of the preferredembodiments of the invention with reference to the drawings, in which:

FIG. 1 is a line graph showing PHB accumulation versus time for avariety of E. coli clones containing different plasmid constructs;

FIGS. 2a and 2b are bar graphs showing the accumulation of PHB producedby transformed E. coli using minimal medium and whey;

FIG. 3 is a bar graph showing the percentage of PHB agglomeration usingCaCl₂ ;

FIG. 4 is a line graph showing the PHB agglomerations versus time wherePHB is accumulated in the presence of radiolabelled glucose and thensubjected to the agglomeration procedure; and

FIG. 5 is a bar graph showing the contrasting effects of glass milk andcalcium on PHB agglomeration.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION

Referring now to the drawings, and more particularly to FIG. 1, it isshown that the E. coli strain HMS174 containing the plasmid p4Aaccumulates a greater percentage of PHB in a shorter period of time thanother E. coli clones containing different plasmid constructs. The E.coli strain HMS174 is available from the Yale E. coli Stock Center,Dept. of Biology, 355 OML, Yale University, P.O. Box 6666, New Haven,Conn. 06511-7444, Barbara Bachman curator. The p4A plasmid carries thePHB biosynthetic pathway and approximately four hundred extra bases tothe upstream and downstream sides of the PHB biosynthetic pathway on thevector pTZ-18U. The vector pTZ-18U is available from United StatesBiochemicals. MSA carries the PHB biosynthetic pathway on the vectorpTZ-18U and the E-lysis gene from phage phi X 174 on another compatibleplasmid. MSA differs from p4A in that it has approximately four hundredextra bases on the upstream side of the PHB biosynthetic pathway (i.e.,the PHB biosynthetic pathway is cloned into pTZ-18U to createpTZ-18U--PHB called "MSA", and p4A is pTZ-18U-PHB less four hundredbases on the upstream side of the PHB biosynthetic pathway). GEM carriesthe PHB biosynthetic pathway on the vector pGEM-7F+ which is availablefrom the Promega Corporation.

The p4A, pTZ-18U--PHB (MSA), and pGEM7f-PHB(GEM) clones were allconstructed from the E. coli clone harboring the PHB biosyntheticpathway discussed in the above-referenced and incorporated co-pendingpatent application and journal article using conventional molecularcloning techniques. As was disclosed in the patent application andjournal article, the PHB biosynthetic pathway can be isolated from A.eutrophusand expressed in E. coli. The PHB biosynthetic pathway isapproximately five kilobases in length and contains bases coding forβ-ketothiolase, NADP-linked acetoacetyl-coenzyme A (CoA) reductase, andPHB synthetase. FIG. 1 shows that the MSA and GEM clones do not produceas much PHB as the p4A clone.

E. coli HMS174 was chosen as the host because it contains a lactoseutilization system and it is recombination deficient. Recombinationdeficiency assures that a plasmid containing lactose genetic regionswill not recombine and make the construct unstable. As will be describedbelow, the presence of the lactose utilization system in HMS174 allowswhey, a cheese manufacturing waste product whose major component islactose, to be used as the carbon source for PHB production. In makingthe transformed E. coli strain, the plasmid p4A, which is the PHBbiosynthetic pathway plus four hundred bases upstream and downstream ofthe PHB biosynthetic pathway cloned into the United States Biochemicalvector pTZ-18U, is electroplated into the E. coli HMS174. A strain ofthe E. coli harboring the p4A plasmid has been deposited with theAmerican Type Culture Collection of 12301 Parklawn Drive, Rockville, Md.on May 23, 1990, and bears deposit number: 68329 designated as E. coliHMS174/p4A (PHB 90) and deposited in accordance with the requirements ofthe Budapest Treaty.

Experiments were performed which showed that the HMS174 strain of E.coli which had been transformed with the p4A plasmid could be grown onminimal medium containing whey. The minimal medium used was M9 minimalmedium which is described in most microbiological biology texts. Table 1lists the formulation for a 5X concentrate of M9 minimal medium whereeach of the listed components is added to a liter flask and water isadded to one liter.

                  TABLE 1                                                         ______________________________________                                        5X M9 MINIMAL MEDIUM FORMULATION                                              ______________________________________                                                   30 g Na.sub.2 HPO.sub.4                                                       15 g KH.sub.2 PO.sub.4                                                        5 g  NH.sub.4 Cl                                                              2.5 g                                                                              NaCl                                                          ______________________________________                                    

Whey was purchased from Sigma chemicals as a powder of bovine whey, andwas made by stirring 20 grams of whey in water that had a final volumeof 100 ml. Stirring took place with mild heating for approximately 30minutes. This solution was then autoclaved and particulates thatprecipitated during centrifugation were pelleted by centrifugation at10,000×g for 10 min. The remaining supernate was used as the whey carbonsource.

In the experiments, the HMS174 E. coli strain containing the plasmid p4Awas inoculated from a plate culture into 50 ml of M9 minimal medium+wheysolution. Table 2 lists the formulation for 50 ml of minimal mediumcontaining whey at a final concentration of 8%.

                  TABLE 2                                                         ______________________________________                                        MINIMAL MEDIUM + WHEY FOR PHB PRODUCTION                                      ______________________________________                                        10        ml    5X M9 medium                                                  20        ml    ddH.sub.2 O (double distilled water)                          50        μl 1 M MgSO.sub.4                                                5         μl 0.5% Thiamine                                                 250       μl 20% casamino acids                                            20        ml    20% whey solution                                             ______________________________________                                    

The inoculated culture was grown at 37° C. for 48 hours in an orbitalincubator shaker at 300 rpm in a 250 ml baffled flask. After the 48 hourincubation time, the culture was stopped and the cells were harvested.Gas chromatography was used to analyze the PHB content.

FIGS. 2a and 2b respectively show the percentage of PHB accumulated inthe cells, expressed as PHB weight per cell divided by the total weightof the cell, and the yield of PHB in the cells, expressed as the totalPHB made in mg/ml, for differing concentrations of whey in solution withthe minimal medium. FIGS. 2a and 2b show that even with very lowconcentrations of whey, i.e., 2% in solution, high concentrations of PHBaccumulation (i.e., greater than 90%) and high yields of PHB (i.e.,approximately 10 mg/ml). While FIGS. 2a and 2b show that medium withhigher concentrations of whey tended to produce greater concentrationsand yields of PHB, it was noted that after the whey concentrationexceeds 8%, PHB production begins to fall.

In the above experiments, PHB production was analyzed after forty eighthours of incubation; however, it should be noted that significant PHBproduction was observed after twenty four hours of incubation. Inaddition, it is anticipated that the relative concentrations of the Na₂HPO₄, KH₂ PO₄, NH₄ Cl , and NaCl in the 5X minimal medium formulationand the relative concentrations of the 5X minimal medium, doubledistilled water, MgSO₄, Thiamine, casamino acids, and whey solutioncould be varied while still allowing production of PHB in a transformedE. coli host having a lactose utilization system.

Utilizing whey as the carbon source for the production of PHB, where thewhey is present in minimal medium, is expected to result in considerablecost savings over the prior art practice of using rich medium withglucose for producing PHB. The prior art transformed E. coli cells whichhad plasmids coding for the PHB biosynthetic pathway, which werediscussed in the co-pending patent application and journal article,could not be grown using whey as the carbon source since those bacteriadid not have a lactose utilization system present therein. PHB cannot beproduced using whey as its carbon source in the native host, Alcaligeneseutrophus, because that bacteria also lacks a lactose utilizationsystem. In addition, as shown in FIG. 1, transforming a particular E.coli host, HMS174, with a particular plasmid, p4A, allows the productionof PHB at much higher percentages than when the E. coli is transformedwith a different vector which also codes for the PHB biosyntheticpathway.

Because the PHB is being produced in E. coli, rather than its nativehost (A. eutrophus), the applicant believed that the PHB polymerproduced by the transformed E. coli might have different physicalproperties from PHB produced in A. eutrophus. In particular, theapplicant conducted experiments to determine if PHB produced by atransformed E. coli could be agglomerated by various ionic solutions. Inthe experiments, PHB was produced in transformed E. coli as discussed inthe above-incorporated co-pending patent application and journalarticle. Briefly, a PHB-producing strain is grown in Luria broth (LB)containing 1% glucose for 24 hours at 37° C. in a shake flask culture.The cells are pelleted by centrifugation (2,000×g for 5 min) and thenresuspended in a volume of water equal to the original culture. Thecells were then lysed by sonication and various ionic reagents wereadded to the solution. Table 3 shows the aggregative effect on PHBproduced in transformed E. coli by various ionic solutions.

                  TABLE 3                                                         ______________________________________                                        AGGREGATION OF                                                                PHB BY VARIOUS IONIC SOLUTIONS                                                Solution*       Degree of Aggregation**                                       ______________________________________                                        KH.sub.2 PO.sub.4                                                                             ++                                                            NaCl            +                                                             CsCl            -                                                             MgSO.sub.4      +++                                                           K.sub.2 HPO.sub.4                                                                             +                                                             MgCl.sub.2      ++++                                                          (NH.sub.4).sub.2 HPO.sub.4                                                                    +                                                             MgOAc           ++++                                                          NaOAc           ++                                                            KCl             -                                                             KOAc            -                                                             CaCl.sub.2      ++++                                                          (NH.sub.4)OAc   -                                                             ______________________________________                                         *All solutions were at a final concentration of 1M.                           **Agglomeration was subjectively graded using micrographs of each             aggregate. "++++" signifies the best agglomeration and "+" signifies the      lowest amount of agglomeration. "-" signifies no agglomeration.          

Table 3 shows that several ionic solutions cause PHB produced intransformed E. coli to agglomerate. The best agglomerating agent wasCaCl₂ based on a subjective judgement concerning the speed and size ofthe agglomerates. The agglomeration effect of CaCl₂ on PHB produced intransformed E. coli is especially interesting since CaCl₂ does not causePHB produced in its native A. eutrophus to agglomerate (i.e., anexperiment was performed where PHB granules were obtained from lysedAlcaligenes H16 eutrophus and subjected to calcium chloride wherein noagglomeration was observed).

Experiments were conducted to determine the ideal concentration of CaCl₂to use for agglomerating PHB. In the experiments, the transformed E.coli cells were prepared and lysed as described above, then thesolutions were brought to different mM CaCl₂ concentrations using a 1Mstock CaCl₂ solution. With low concentrations of CaCl₂, e.g., 1 mM, verylong incubation times were required for PHB granules to agglomerate andonly small agglomerates were produced. With high concentrations ofCaCl₂, e.g., 100 mM and above, agglomeration occurred almostinstantaneously and resulted in large "snowflake"-like particles thatfell to the bottom of the tube. However, the agglomerates achieved withhigh concentrations of CaCl₂ appeared to have large amounts of celldebris. Therefore, high concentrations of CaCl₂ are not desirable foragglomeration. When medium concentrations of CaCl₂ were used, e.g., 5 mMto 30 mM, agglomeration of medium sized pellets occurred within a shortincubation period of 5 to 15 minutes. Use of 10 mM CaCl₂ was determinedto produce the best agglomeration results in terms of speed and size ofagglomerate formation.

Experiments were performed to determine the percentage of PHBagglomerated by CaCl₂ versus the percentage of PHB left in solution. Inthe experiments, a PHB-producing strain of E. coli was grown in Luriabroth containing 1% radio labelled glucose for 24 hours at 37° C. in ashake flask culture. The cells were pelleted by centrifugation (2,000×gfor 5 min) and then resuspended in a volume of water equal to theoriginal culture. The cells were then lysed by sonication and then thesolution was brought to 10 mM by the addition of a 1M calcium chloridestock. The tube was incubated 10 min at room temperature and thencentrifuged at 400×g for 2 min. The agglomerated PHB granules pelleted,while much of the cell debris stayed in the supernate. The supernatantwas then aspirated. To determine the distribution of PHB in the pelletand supernatant, the pellet and supernatant were measured using eithercapillary gas chromatography or liquid scintillation counting.

FIG. 3 shows that nearly all (100%) of the PHB in the culture wasagglomerated and recovered by the above process. In this experiment, theamount of PHB was measured only by gas capillary chromatography. Thisexperiment was done at several cell volumes to determine if the volumeof the flask influenced the degree of agglomeration and it was foundthat in all volumes nearly all of the PHB was agglomerated andsubsequently pelleted by centrifugation.

FIG. 4 shows that it is extremely important that the culture be allowedsufficient time for agglomeration to occur, otherwise the yield isreduced. Rather than allowing a full ten minute incubation time afterthe solution was adjusted to 10 mM CaCl₂, the pellet and supernatantfractions were counted at two minute timed intervals after theadjustment. FIG. 4 shows that during the first few minutes after addingCaCl₂ the amount of PHB present in the supernatant is actually greaterthan in the pellet. However, after eight minutes (where the amount ofPHB measured in the pellet begins to level off), the amount of PHB inthe pellet is far greater than in the supernatant fraction. It should benoted at this point that this experiment measures radioactive ¹⁴ Carbon,most of which is incorporated into PHB as ¹⁴ C-glucose (approximately60% is incorporated), but some of which is present as soluble material.Therefore, even though nearly all of the PHB is precipitated, there isstill a large number of counts in the superhate that is due to thesoluble radioactive glucose.

FIG. 5 shows that agglomeration of PHB can be enhanced by the additionof nucleating agents such as glass milk available from Bio 101. In FIG.5, the counts per minute (CPM) of the pellet and supernatant fractionsare displayed where "+gm, +Ca" indicates PHB agglomeration in thepresence of glass milk and 10 mM CaCl₂, "-gm" indicates PHBagglomeration in the presence of 10 mM CaCl₂ without glass milk, "-gm,-Ca" indicates PHB agglomeration in the absence of glass milk and CaCl₂,and "-Ca" indicates PHB agglomeration in the presence of glass milk andin the absence of CaCl₂. From FIG. 5, it can be seen that theenhancement of agglomeration by the addition of nucleating agents is notvery large; therefore, larger production schemes may not be greatlybenefitted by the use of such agents.

While the invention has been described in terms of its preferredembodiments where a strain of transformed E. coli has been created whichcan accumulate larger quantities of PHB while using an inexpensivecarbon source such as whey for PHB production and an ionic solution suchas CaCl₂ can be used to agglomerate PHB, those skilled in the art willrecognize that the invention can be practiced with modification withinthe spirit and scope of the appended claims.

Having thus described my invention, what I claim as new and desire tosecure by Letters Patent is as follows:
 1. An Escherichia coli bacterialhost transformed by a vector consisting essentially of adeoxyribonucleic acid sequence encoding the poly-beta-hydroxybutyratebiosynthetic pathway of Alcaligenes eutrophus wherein the bacterial hosthas a lactose utilization system and grows in minimal medium containingwhey and wherein the bacterial host is Escherichia coli strain ATCC68329.
 2. A method for producing poly-beta-hydroxybutrate, comprisingthe steps of:providing a culture of the bacterial host of claim 1;growing said culture in a growth medium comprising minimal medium andwhey for a period greater than twenty four hours, said bacterial hostproducing intra-cellular poly-beta-hydroxybutyrate; lysing saidbacterial host in said culture to release said poly-betahydroxybutyrate;and collecting said poly-beta-hydroxybutyrate.
 3. The method as recitedin claim 2 in which said Escherichia coli bacterial host is grown in agrowth medium comprising:
 0. 6% Na₂ HPO₄ ;0.3% KH₂ PO₄ ; 0.1% ammoniumchloride; 0.05% sodium chloride; 58.84% water; 0.012% magnesium sulfate;0.0005% thiamine; 0.01% casamino acids; and, 40% whey solution.
 4. Themethod as recited in claim 2 wherein said minimal medium isapproximately twenty percent of said growth medium, said whey isapproximately forty percent of said growth medium, and water isapproximately forty percent of said growth medium.
 5. A method asrecited in claim 2 wherein said step of collecting includes the step ofexposing said released poly-beta-hydroxybutyrate to an ionic reagentselected from the group consisting of magnesium sulfate, magnesiumchloride, magnesium acetate, and calcium chloride, said ionic reagentbeing of sufficient concentration to agglomerate saidpoly-beta-hydroxybutyrate.
 6. A method as recited in claim 5 whereinsaid ionic reagent is calcium chloride at a concentration rangingbetween one molar and one millimolar.
 7. A method as recited in claim 6wherein said calcium chloride has a concentration of approximately tenmillimolar.
 8. A method for agglomerating poly-beta-hydroxybutyratewhich has been intracellularly produced in a culture of Escherichia coli(strain ATCC 68329) bacterial host which has been transformed by avector the vector containing a deoxyribonucleic acid sequence coding forthe poly-beta-hydroxybutyrate biosynthetic pathway from Alcaligeneseutrophus, comprising the steps of:lysing said bacterial hosts torelease said poly-beta-hydroxybutyrate; and adding to said releasedpoly-beta-hydroxybutyrate a sufficient quantity of an ionic reagentselected from the group consisting of magnesium sulfate, magnesiumchloride, magnesium acetate and calcium chloride, said sufficientquantity of said ionic reagent agglomerating granules of saidpoly-beta-hydroxybutyrate.
 9. A method as recited in claim 8 whereinsaid ionic reagent is calcium chloride.
 10. A method as recited in claim8 wherein said calcium chloride is present at a concentration rangingbetween one molar and one millimolar.
 11. A method as recited in claim10 wherein said calcium chloride is present at a concentration ofapproximately 10 mM.
 12. A plasmid designated as p4A and deposited withthe American Type Culture Collection in Escherichia coli strain HMS 174under accession number
 68329. 13. The method recited in claim 8 furthercomprising the step of adding a nucleating agent to said quantity ofsaid ionic reagent.
 14. A method as recited in claim 10 wherein saidcalcium chloride is present at a concentration ranging from about 5 mMto about 30 mM.
 15. A plasmid designated as pTZ-18U PHB and depositedwith the American Type Culture Collection in Escherichia coli strainHMS174 under accession number 69064.